Analysis of the efficiency and efficacy of an in vitro CRISPR prime editing–based correction approach targeting a splice-site mutation in the OPTN gene

Objective: Prime Editing (PE) is a next-generation genome editing approach within CRISPR–Cas-based technologies that enables precise nucleotide modifications with high specificity without inducing double-strand DNA breaks and with reduced off-target effects. Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of motor neurons and limited therapeutic options. ALS type 12 (ALS12) is associated with pathogenic variants in the OPTN gene. In this thesis, we aimed to evaluate the efficiency and effectiveness of in vitro correction of the OPTN c.1242+1G>A splice-site variant using the PE2 system in patient-derived primary fibroblast cells. Materials and Methods: An original epegRNA targeting the OPTN c.1242+1G>A variant was designed in silico. The epegRNA sequences were cloned into the pU6-GG-acceptor vector using the Golden Gate cloning method and co-transfected with the Cas9 nickase–reverse transcriptase–containing pCMV-PEmax plasmid into patient-derived primary fibroblast cells via Lipofectamine-mediated transfection. In addition, the use of OPTN:c.1242+1G>A_eGFP-epegRNA and pCMV-PE2-tagRFP-BleoR plasmids enabled validation of transfection efficiency through GFP and RFP fluorescent markers. Following Prime Editing, genomic DNA was isolated, the target region was amplified, and next-generation sequencing (NGS) was performed. Editing efficiency was calculated by quantitative analysis of high-depth sequencing data using Integrative Genomics Viewer (IGV). The study was conducted in accordance with institutional ethics committee approval and informed consent obtained from the patient. Results: NGS analysis revealed that the targeted A>G correction at the OPTN:c.1242+1 position was achieved at frequencies ranging from approximately 0.07% to 0.41% across different transfection pools. Comparable editing rates observed in biologically duplicated experimental pools demonstrated the reproducibility of the results. A positive correlation was identified between increasing plasmid dosage and editing efficiency. The detection of GFP- and RFP-positive cells confirmed successful transfection. Conclusion: This thesis demonstrates for the first time that the OPTN:c.1242+1G>A splice-site variant can be targeted using Prime Editing in patient-derived primary fibroblast cells. Although the observed editing efficiencies were relatively low, the findings highlight the feasibility of applying PE technology in patient-specific cellular models for neurodegenerative diseases such as ALS. Importantly, this correction holds potential therapeutic relevance by enabling restoration of the normal splicing mechanism of the OPTN gene. The results suggest that further optimization of Prime Editing strategies and advanced cellular selection approaches may facilitate translation toward clinical applications.